The long-term objective for the research described in this application is directed at a comprehensive understanding of the physical and chemical parameters that relate protein structure and substrate recognition in enzyme-catalyzed reactions. This objective is aimed toward the development of general methodologies and novel protocols for the determination of reaction and substrate specificities for enzymes of unknown function. A critical assessment of the functional annotations forthe more than four million genes that have been sequenced to date suggests that approximately one-third of the encoded proteins have an uncertain, unknown, or /ncorrecf functional assignment. This observation suggests that a significant fraction of the metabolic diversity remains to be properly characterized. Toward this end we will utilize computational docking of high energy intermediates to models of the active sites for enzymes of unknown function to identify the most probable substrates. These efforts will be complemented by the synthesis and screening of chemical libraries and the abstraction of further metabolic information from operon and genomic context. This goal will be pursued by concentrating on the elucidation of the substrate and reaction profiles forthe entire ensemble of enzymes within the amidohydrolase superfamily. The amidohydrolase superfamily is a group of enzymes which has a substantial substrate diversity embedded within active sites that are forged from a (p/a)8-barrel structural fold. Over 6,000 unique protein sequences have been identified as members of the amidohydrolase superfamily. Members of this superfamily have been shown to catalyze the hydrolysis of amides, lactones and organophosphate esters, in addition to decarboxylation, hydration, and isomerization reactions. However, a substantial fraction of the members of this broad superfamily have an ambiguous substrate and reaction specificity that remains to be unraveled. Members of this superfamily of enzymes include dihydroorotase, urease, and adenosine deaminase. The hallmark for this particular superfamily of enzymes is an active site at the C-terminal end of a (p/a)8-barrel structural domain that contains a mononuclear or binuclear metal center that functions predominantly, but not exclusively, to activate solvent water for nucleophilic attack on electrophilic functional groups. The substrate and reaction diversity contained within this enzyme superfamily will provide unique insights into the molecular mechanisms for the evolution and development of novel enzymatic activities from existing structural templates. RELEVANCE (See instructions); The overall objective of this application is directed towards the development of novel and general methods for the elucidation of function for enzymes with unknown substrates. These efforts will unveil new metabolic transformations and identify new targets for therapeutic intervention.